The mechanisms by which hormones regulate retinal vascular circulation are not clear. Recently, a new and potent vasoactive hormone, endothelin, has been described and sequenced from vascular endothelial cells. The endothelin (ET) family consists of at least three hormones which have a vasoconstrictive effect on vascular smooth muscle cells. Recently, evidence has indicated that ET probably has a role in regulating retinal circulation since it has been identified in the retina and retinal vascular cells. Since retinal capillary cells have high affinity and specific receptor for ET-1, ET injected into rabbit vitreous was shown to induce vasoconstriction of retinal vessels. We have recently shown that the expression of ET-1 mRNA is regulated by insulin via its own receptor through a novel mechanism. In addition, we have shown that retinal pericytes have ET-1 receptors of 95 and 65 kD whereas smooth muscle cells only have the 60 kD receptor. Lastly, using image analysis of video fluorescein angiogram, the intravitreal bolus injection of 1 x 10 19 M of ET-1 induced vasoconstriction of retinal arteries and arterioles and increased retinal circulation times for as long as 20 min after infusion. We are proposing to study the following three Specific Aims: la) to determine the regulation of ET-1 in retinal and aortic endothelial cells by insulin at the DNA level; b) to characterize the role of high glucose level and protein kinase C in regulating ET-1 expression; c) to correlate the expression of ET-1 in the retina, aorta and kidney with vascular abnormalities in diabetic rats. 2a) To characterize, purify and sequence the receptors of ET-1 in retinal pericytes; b) to determine the biological effects of ET-1 in pericytes on the stimulation or activation of diacylglycerol, IP3, protein kinase C and calponin. 3) To evaluate the physiological actions of ET on retinal circulation in diabetic and non-diabetic rats. From these studies, we will be able to understand ET's expression and mode of action at the genetic, biochemical, cellular and physiological levels with special focus on its role in the development of retinal vascular dysfunctions in diabetes.